DESCRIPTION: The primer for reverse transcription in HIV-1, human tRNALys3, is selectively packaged into virions along with tRNALys1,2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNALys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors is required for optimal viral infectivity. LysR is normally part of a dynamic mammalian multisynthetase complex (MSC). In recent years, LysRS has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways. Elucidating the detailed molecular mechanism for the alternative function of LysRS in HIV-1 infectivity is needed in order to develop effective therapeutics aimed at targeting this essential host cell factor. While some aspects of tRNA primer packaging into HIV-1 particles are now understood, the mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into particles through interaction with Gag is unclear, and this is the key question to be addressed in Aim 1. We have recently shown that LysRS binds with high affinity to the primer binding site region in the 5'UTR of HIV-1 genomic RNA (vRNA). In preliminary studies, we found that this highly structured and dynamic region of the genome contains a tRNA-like element that is responsible for the interaction with LysRS. However, the conformational changes that occur upon LysRS binding, tRNA primer annealing, and subsequent binding and initiation by reverse transcriptase are unknown, and no high-resolution structure of the initiation complex has been reported to date. These key open questions will be addressed in Aims 2 and 3, respectively. The specific aims of the proposed work are: (1) To elucidate the mechanism by which human LysRS is recruited into HIV-1 particles, (2) To characterize the global tertiary structure and conformational changes of the PBS region of HIV-1 vRNA upon LysRS binding and tRNA primer annealing, and (3) To elucidate the structure of the vRNA:primer:RT initiation complex.